Favorable Coulombic Interactions Research Articles

Self-Assembly of Insulin-Derived Chimeric Peptides into Two-Component Amyloid Fibrils: The Role of Coulombic Interactions.

Canonical amyloid fibrils are composed of covalently identical polypeptide chains. Here, we employ kinetic assays, atomic force microscopy, infrared spectroscopy, circular dichroism, and molecular dynamics simulations to study fibrillization patterns of two chimeric peptides, ACC1-13E8 and ACC1-13K8, in which a potent amyloidogenic stretch derived from the N-terminal segment of the insulin A-chain (ACC1-13) is coupled to octaglutamate or octalysine segments, respectively. While large electric charges prevent aggregation of either peptide at neutral pH, stoichiometric mixing of ACC1-13E8 and ACC1-13K8 triggers rapid self-assembly of two-component fibrils driven by favorable Coulombic interactions. The low-symmetry nonpolar ACC1-13 pilot sequence is crucial in enforcing the fibrillar structure consisting of parallel β-sheets as the self-assembly of free poly-E and poly-K chains under similar conditions results in amorphous antiparallel β-sheets. Interestingly, ACC1-13E8 forms highly ordered fibrils also when paired with nonpolypeptide polycationic amines such as branched polyethylenimine, instead of ACC1-13K8. Such synthetic polycations are more effective in triggering the fibrillization of ACC1-13E8 than poly-K (or poly-E in the case of ACC1-13K8). The high conformational flexibility of these polyamines makes up for the apparent mismatch in periodicity of charged groups. The results are discussed in the context of mechanisms of heterogeneous disease-related amyloidogenesis.

Photocatalytic Regeneration of a Nicotinamide Adenine Nucleotide Mimic with Water-Soluble Iridium(III) Complexes.

Nicotinamide adenine nucleotide (NADH) is involved in many biologically relevant redox reactions, and the photochemical regeneration of its oxidized form (NAD+) under physiological conditions is of interest for combined photo- and biocatalysis. Here, we demonstrate that tri-anionic, water-soluble variants of typically very lipophilic iridium(III) complexes can photo-catalyze the reduction of an NAD+ mimic in a comparatively efficient manner. In combination with a well-known rhodium co-catalyst to facilitate regioselective reactions, these iridium(III) photo-reductants outcompete the commonly used [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) photosensitizer in water by up to 1 order of magnitude in turnover frequency. This improved reactivity is attributable to the strong excited-state electron donor properties and the good chemical robustness of the tri-anionic iridium(III) sensitizers, combined with their favorable Coulombic interaction with the di-cationic rhodium co-catalyst. Our findings seem relevant in the greater context of photobiocatalysis, for which access to strong, efficient, and robust photoreductants with good water solubility can be essential.

Catalysis of Hydrogen-Deuterium Exchange Reactions by 4-Substituted Proline Derivatives.

The identification and understanding of structure-activity relationships is vital for rational catalyst design. A kinetic study of the hydrogen-deuterium exchange reaction of cyclohexanone in aqueous solution, as catalyzed by proline derivatives, has revealed valuable structure-activity relationships. In phosphate-buffered solution, cis-4-fluoroproline is more active than the trans isomer, a distinction that appears to originate from a destabilizing interaction between the fluorine atom and phosphate anion during general acid-catalyzed dehydration of the carbinolamine intermediate. trans-4-Ammoniumprolines are exceptionally active catalysts owing to favorable Coulombic interactions involving the ammonium group and the alkoxide moiety formed upon 1,2-addition of the proline derivative to the ketone. These results could be used for the optimization of proline catalysts, especially in transformations where the formation of the putative iminium ion is rate-limiting.

Bridging Calorimetry and Simulation through Precise Calculations of Cucurbituril-Guest Binding Enthalpies.

We used microsecond time scale molecular dynamics simulations to compute, at high precision, binding enthalpies for cucurbit[7]uril (CB7) with eight guests in aqueous solution. The results correlate well with experimental data from previously published isothermal titration calorimetry studies, and decomposition of the computed binding enthalpies by interaction type provides plausible mechanistic insights. Thus, dispersion interactions appear to play a key role in stabilizing these complexes, due at least in part to the fact that their packing density is greater than that of water. On the other hand, strongly favorable Coulombic interactions between the host and guests are compensated by unfavorable solvent contributions, leaving relatively modest electrostatic contributions to the binding enthalpies. The better steric fit of the aliphatic guests into the circular host appears to explain why their binding enthalpies tend to be more favorable than those of the more planar aromatic guests. The present calculations also bear on the validity of the simulation force field. Somewhat unexpectedly, the TIP3P water yields better agreement with experiment than the TIP4P-Ew water model, although the latter is known to replicate the properties of pure water more accurately. More broadly, the present results demonstrate the potential for computational calorimetry to provide atomistic explanations for thermodynamic observations.

Conformational Effects in Non-Stoichiometric Complexes of Two Hyperbranched Molecules with a Linear Polyelectrolyte

We report results from Brownian dynamics computer simulations of systems comprised by two terminally charged hyperbranched molecules preferentially branched in the periphery, with an oppositely charged linear chain of varying length. Comparison of the findings from the present study to stoichiometric counterparts and to analogous dendrimer-based complexes, reveal that the presence of the second hyperbranched molecule incurs significant changes in the conformational characteristics of both components of the complex. Instead of step-like changes in the average size and shape of the hyperbranched component that were noted in the previously studied stoichiometric systems, a rather smooth change is observed upon increase of the length of the linear component. In addition, a markedly different behavior is also noticed in the conformational characteristics of the linear chain when compared to that in similar dendrimer-based systems. The above findings are consistent with the higher degree of deformability of the peripherally branched molecules which allow appropriate rearrangements in shape in order to accommodate the favorable Coulombic interactions between the two components of the complex. This behavior offers new insight towards the design of more efficient hyperbranched-based systems which can take advantage of the multifunctionality and the structural properties of the highly branched polymer components.

Supramolecular Chirality in Solvent‐Promoted Aggregation of Amphiphilic Porphyrin Derivatives: Kinetic Studies and Comparison between Solution Behavior and Solid‐State Morphology by AFM Topography

The solvent-promoted aggregation behavior of some amphiphilic porphyrin derivatives bearing chiral functionality in the form of a charged L-proline group has been investigated by UV/Vis, resonance light scattering, fluorescence and circular dichroism spectroscopy. The investigated macrocycles give rise to aggregates featuring supramolecular chirality with high ellipticity. Kinetic studies reveal peculiar differences in the fashion of aggregation, depending on the intimate nature of the chiral functionality, namely, cationic (nitrogen-quaternized L-proline, 3H(2)) or anionic (carboxylate residue, 6H(2)) group. Formation of anionic 6H(2) aggregates shows a diffusion-limited kinetic behavior. AFM topography studies show formation of tighter globular structures. On the other hand, the corresponding 3H(2) aggregates are formed by a cooperative, fractal-type decay, and appear as long-fibrous, looser structures. In the templated aggregation of 3H(2) over preformed 6H(2) aggregates, AFM images show formation of globular structures with reduced sizes, as a likely consequence of shorter interchromophore distances, due to favorable Coulombic interactions. The results obtained show an interesting parallelism between the solution behavior and the solid-state aggregate structures, corroborating the sergeant-soldier effect observed in the templated aggregation. The results presented give important insights for understanding the complex mechanisms involved in these issues, which are of key importance for the development of chiral supramolecular materials and stereoselective sensors and devices.

Terminal sidechain packing of a designed β‐hairpin influences conformation and stability

While end capping in alpha-helices is well understood, the concept of capping a beta-hairpin is a relatively recent development; to date, favorable Coulombic interactions are the only example of sidechains at the termini influencing the overall stability of a beta-hairpin. While cross-strand hydrophobic residues generally provide hairpin stabilization, particular when flanking the turn region, those remote from this location appear to provide little stabilization. While probing for an optimal residue at a hydrogen bond position near the terminus of a designed beta-hairpin a conservative, hydrophobic, V --> I mutation was observed to not only result in a significant change in fold population but also effected major changes in the structuring shifts at numerous sites in the peptide. Mutational studies reveal that there is an interaction between the sidechain at this H-bonded site and the sidechain at the C-terminal non-H-bonded site of the hairpin. This interaction, which appears to be hydrophobic in character, requires a highly twisted hairpin structure. Modifications at the C-terminal site, for example an E --> A mutation (DeltaDeltaG(U) = 6 kJ/mol), have profound affects on fold structure and stability. The data suggests that this may be a case of hairpin end capping by the formation of a hydrophobic cluster.

Theory-guided design of Brønsted acid-assisted phosphine catalysis: synthesis of dihydropyrones from aldehydes and allenoates

The phosphine-catalyzed addition of 2,3-butadienoates to aldehydes has been extended to the formation of disubstituted dihydro-2-pyrones. The requisite shift in equilibrium of the intermediate zwitterionic β-phosphonium dienolates toward the s- cis intermediate was accomplished through the use of a Brønsted acid additive, which disrupts the favorable Coulombic interaction present in the s- trans intermediate. The detailed nature of the synergistic interactions involving the Brønsted acid additives and phosphine involved in the formation of s- cis β-phosphonium dienolates was analyzed through a series of DFT calculations. Unlike previously reported annulations of aldehydes with allenoates, where trialkylphosphines are optimal catalysts, in this study triphenylphosphine was also found for the first time to be a suitable catalyst for the synthesis of dihydropyrones. This method provides a one-step route toward functionalized dihydropyrones from simple, stable starting materials. In addition, new reaction pathways of phosphine-catalyzed allene annulations are unveiled, with the formation of dihydropyrones being the first example of dual activation in this sphere.

Structural Basis for Catalysis by Onconase

Onconase® (ONC) is a homolog of bovine pancreatic ribonuclease (RNase A) from the frog Rana pipiens. ONC displays antitumoral activity and is in advanced clinical trials for the treatment of cancer. Here, we report the first atomic structures of ONC–nucleic acid complexes: a T89N/E91A ONC–5′-AMP complex at 1.65 Å resolution and a wild-type ONC–d(AUGA) complex at 1.90 Å resolution. The latter structure and site-directed mutagenesis were used to reveal the atomic basis for substrate recognition and turnover by ONC. The residues in ONC that are proximal to the scissile phosphodiester bond (His10, Lys31, and His97) and uracil nucleobase (Thr35, Asp67, and Phe98) are conserved from RNase A and serve to generate a similar bell-shaped pH versus k cat/ K M profile for RNA cleavage. Glu91 of ONC forms two hydrogen bonds with the guanine nucleobase in d(AUGA), and Thr89 is in close proximity to that nucleobase. Installing a neutral or cationic residue at position 91 or an asparagine residue at position 89 virtually eliminated the 10 2-fold guanine:adenine preference of ONC. A variant that combined such substitutions, T89N/E91A ONC, actually preferred adenine over guanine. In contrast, installing an arginine residue at position 91 increased the guanine preference and afforded an ONC variant with the highest known k cat/ K M value. These data indicate that ONC discriminates between guanine and adenine by using Coulombic interactions and a network of hydrogen bonds. The structure of the ONC–d(AUGA) complex was also used to probe other aspects of catalysis. For example, the T5R substitution, designed to create a favorable Coulombic interaction between ONC and a phosphoryl group in RNA, increased ribonucleolytic activity by twofold. No variant, however, was more toxic to human cancer cells than wild-type ONC. Together, these findings provide a cynosure for understanding catalysis of RNA cleavage in a system of high medicinal relevance.

Mechanism and Diastereoselectivity of Aziridine Formation from Sulfur Ylides and Imines: A Computational Study

A computational investigation of the title reaction involving semistabilized (R = Ph) and stabilized (R = CO2Me) sulfur ylides has been performed using DFT methods including a continuum model of solvent. Our results provide support for the generally accepted mechanism and are in very good agreement with observed cis/trans selectivities. This study shows that betaine formation is nonreversible, and that selectivity is thereby determined at the initial addition step, in the case of semistabilized ylides. Our analysis indicates moreover that addition TS structures are governed by the steric strain induced by the N-sulfonyl group, which favors the transoid approach in the case of syn betaine formation and the cisoid mode of addition in anti TSs. The observed low trans selectivity is accounted for by the favorable Coulombic interactions and stabilization by C-H...O hydrogen bonding allowed in the cisoid anti addition TS. In the case of stabilized ylides, the endothermicity of betaine formation combined with the high barrier to ring closure render the elimination step rate- and selectivity-determining. Accordingly, the low cis selectivity observed in stabilized ylide reactions is explained by the lower steric strain in the elimination step generated by the formation of the cis aziridine (as compared to the trans case).

Enhanced hairpin stability through loop design: the case of the protein G B1 domain hairpin.

A mutational study of the peptide corresponding to the second hairpin of the protein G B1 domain (GB1p) provided a series of mutants with significantly increased fold stability. Mutations focused on improvement of the direction-reversing loop and the addition of favorable Coulombic interactions at the sequence termini. The loop optimization was based on a database search for residues that occur with the greatest probability in similar hairpin loops in proteins. This search suggested replacing the native DDATKT sequence with NPATGK, which resulted in a 4.5 kJ/mol stabilization of the hairpin fold. The introduction of positively charged lysines at the N-terminus provided an additional 2.4 kJ/mol of stabilization, affording a GB1p mutant that is 86 +/- 3% folded at 25 degrees C with a melting temperature of 60 +/- 2 degrees C. The trpzip version of this peptide, in which three of the hydrophobic core residues were mutated to tryptophan, yielded a sequence that melted at 85 degrees C. Throughout, fold populations and melting temperatures were derived from the mutation and temperature dependence of proton chemical shifts and were corroborated by circular dichroism (CD) melts. The study also suggests that the wild-type GB1p sequence is significantly less stable than reported in some other studies: only 30% folded in water at 25 degrees C.

A molecular basis for the selectivity of thiadiazole urea inhibitors with stromelysin-1 and gelatinase-A from generalized born molecular dynamics simulations.

Matrix metalloproteinases (MMPs) represent a potentially important class of therapeutic targets for the treatment of diseases such as cancer. Selective inhibition of MMPs will be required given the high sequence identity across the family and the discovery that individual MMPs also regulate the natural angiogenesis inhibitor angiostatin. In this study, we have used computational methods to model the selectivity for six thiadiazole urea inhibitors with stromelysin-1 and gelatinase-A, two homologous MMPs that have been implicated in breast cancer. From continuum Generalized Born molecular dynamics (GB-MD) and MM-GBSA analysis, we estimated ligand free energies of binding using 200 snapshots obtained from a short 40 ps simulation of the relevant protein-ligand complex. The MM-GBSA free energies, computed from the continuum GB-MD trajectories, show strong correlation with the experimental affinities (r(2) = 0.74); prior studies have employed explicit water MD simulations. Including estimates for changes in solute entropy in the binding calculations slightly diminishes the overall correlation with experiment (r2 = 0.71). Notably, in every case, the simulation results correctly predict that a given ligand will bind selectively to stromelysin-1 over gelatinase-A which is gratifying given the high degree of structural homology between the two proteins. The increased selectivity for stromelysin-1 appears to be driven by (1) increased favorable van der Waals interactions, (2) increased favorable Coulombic interactions, and (3) decreased unfavorable total electrostatic energies (Coulombic plus desolvation).

Protein stabilization by salt bridges: concepts, experimental approaches and clarification of some misunderstandings.

Salt bridges in proteins are bonds between oppositely charged residues that are sufficiently close to each other to experience electrostatic attraction. They contribute to protein structure and to the specificity of interaction of proteins with other biomolecules, but in doing so they need not necessarily increase a protein's free energy of unfolding. The net electrostatic free energy of a salt bridge can be partitioned into three components: charge-charge interactions, interactions of charges with permanent dipoles, and desolvation of charges. Energetically favorable Coulombic charge-charge interaction is opposed by often unfavorable desolvation of interacting charges. As a consequence, salt bridges may destabilize the structure of the folded protein. There are two ways to estimate the free energy contribution of salt bridges by experiment: the pK(a) approach and the mutation approach. In the pK(a) approach, the contribution of charges to the free energy of unfolding of a protein is obtained from the change of pK(a) of ionizable groups caused by altered electrostatic interactions upon folding of the protein. The pK(a) approach provides the relative free energy gained or lost when ionizable groups are being charged. In the mutation approach, the coupling free energy between interacting charges is obtained from a double mutant cycle. The coupling free energy is an indirect and approximate measure of the free energy of charge-charge interaction. Neither the pK(a) approach nor the mutation approach can provide the net free energy of a salt bridge. Currently, this is obtained only by computational methods which, however, are often prone to large uncertainties due to simplifying assumptions and insufficient structural information on which calculations are based. This state of affairs makes the precise thermodynamic quantification of salt bridge energies very difficult. This review is focused on concepts and on the assessment of experimental methods and does not cover the vast literature.

Potent Inhibition of Ribonuclease A by Oligo(vinylsulfonic Acid)

Ribonuclease A (RNase A) can make multiple contacts with an RNA substrate. In particular, the enzymatic active site and adjacent subsites bind sequential phosphoryl groups in the RNA backbone through Coulombic interactions. Here, oligomers of vinylsulfonic acid (OVS) are shown to be potent inhibitors of RNase A that exploit these interactions. Inhibition is competitive with substrate and has Ki = 11 pm in assays at low salt concentration. The effect of salt concentration on inhibition indicates that nearly eight favorable Coulombic interactions occur in the RNase A.OVS complex. The phosphonic acid and sulfuric acid analogs of OVS are also potent inhibitors although slightly less effective. OVS is also shown to be a contaminant of MES and other buffers that contain sulfonylethyl groups. Oligomers greater than nine units in length can be isolated from commercial MES buffer. Inhibition by contaminating OVS is responsible for the apparent decrease in catalytic activity that has been observed in assays of RNase A at low salt concentration. Thus, OVS is both a useful inhibitor of RNase A and a potential bane to chemists and biochemists who use ethanesulfonic acid buffers.

Equilibrium and kinetic folding of an alpha-helical Greek key protein domain: caspase recruitment domain (CARD) of RICK.

We have characterized the equilibrium and kinetic folding of a unique protein domain, caspase recruitment domain (CARD), of the RIP-like interacting CLARP kinase (RICK) (RICK-CARD), which adopts a alpha-helical Greek key fold. At equilibrium, the folding of RICK-CARD is well described by a two-state mechanism representing the native and unfolded ensembles. The protein is marginally stable, with a DeltaG(H)()2(O) of 3.0 +/- 0.15 kcal/mol and an m-value of 1.27 +/- 0.06 kcal mol(-1) M(-1) (30 mM Tris-HCl, pH 8, 1 mM DTT, 25 degrees C). While the m-value is constant, the protein stability decreases in the presence of moderate salt concentrations (below 200 mM) and then increases at higher salt concentrations. The results suggest that electrostatic interactions are stabilizing in the native protein, and the favorable Coulombic interactions are reduced at low ionic strength. Above 200 mM salt, the results are consistent with Hofmeister effects. The unfolding pathway of RICK-CARD is complex and contains at least three non-native conformations. The refolding pathway of RICK-CARD also is complex, and the data suggest that the unfolded protein folds via two intermediate conformations prior to reaching the native state. Overall, the data suggest the presence of kinetically trapped, or misfolded, species that are on-pathway both in refolding and in unfolding.

Study of Thermally Treated Lithium Montmorillonite by Ab Initio Methods

The Hofmann−Klemen effect describes the observed reduction in expandable character and cation exchange capacity of lithium-saturated montmorillonite upon mild heating. Although it is widely accepted that the observed changes are due to the lithium ions becoming fixated within the layer structure of the clay mineral, some uncertainty still exists as to the precise location of the migrated lithium ions within the clay framework. It has been suggested that the cations reside in the ditrigonal cavities of the tetrahedral layer, the vacant octahedral sites, or both. We employ density functional methods to examine the phenomenon at the electronic structure level. We find that it is energetically preferable for lithium cations to reside in the vacant octahedral sites as opposed to the ditrigonal cavities due to the closer proximity to the negative charge sites in the octahedral layer increasing favorable Coulombic interactions. Occupation of octahedral sites causes structural hydroxyl groups to reorientate perpe...

Evaluation of the energetic contribution of interhelical coulombic interactions for coiled coil helix orientation specificity

Coiled coils are formed by two or more α-helices that align in a parallel or an antiparallel relative orientation. The factors that determine a preference for a given relative helix orientation are incompletely understood. The helix orientation preference for the designed coiled coil, Acid-a1-Base-a1, was measured previously. This model system therefore provides a means for the experimental determination of the energetic contribution of a variety of interactions to helix orientation specificity. The antiparallel preference for Acid-a1-Base-a1 is imparted by a single buried polar interaction. Interhelical Coulombic interactions between residues at the e and g positions have been proposed to influence helix orientation preference. In the Acid-a1-Base-a1 heterodimer, potentially attractive Coulombic interactions are expected in both orientations. To determine the energetic consequences of Coulombic interactions for helix orientation preference, we have positioned a single charged residue in each peptide such that exclusively favorable interhelical Coulombic interactions can occur only in the parallel orientation. In contrast, two potentially repulsive interactions are expected in the antiparallel orientation. Because the buried polar interaction can occur only in the antiparallel orientation, interhelical Coulombic interactions favor the parallel orientation and the potential to form a buried polar interaction favors the antiparallel orientation. We find no clear preference for an antiparallel orientation in the resulting heterodimer, Acid-K e-Base-E g, suggesting that interhelical Coulombic interactions and a buried polar interaction are of approximately equal importance for helix orientation specificity. Stability measurements indicate that maintenance of all favorable electrostatic interactions and/or avoidance of two potentially repulsive interactions contributes approximately 2.1 kcal/mol to helix orientation preference.

Ester substituted bithiophenes. Abnormally low dihedral angle and rotation barrier due to dipolar stabilization

Ab initio calculations [3−21G (*)] on dimethyl 2, 2′-bithiophene-4, 4′-dicar☐ylate ( 3), dimethyl 2, 2′-bithiophene-3, 4′-dicar☐ylate ( 4) and dimethyl 2, 2′-bithiophene-3, 3′-dicar☐ylate ( 5) have revealed that in 4 there is an abnormally small dihedral angle between the thiophene rings and an abnormally low barrier to rotation around the thiophene-thiophene single bond. This is due to a favorable coulombic interaction of the negative carbonyl oxygen and the positive sulfur of the adjacent ring which helps to flatten and bend the ground state and lower the rotation barrier. Ab inito calculations [3–21G (*)] show a favorable coulombic interaction between the negative carbonyl oxygen and the positive sulfur of the adjacent ring in dimethyl 2, 2′-bithiophene-3, 4′-dicar☐ylate ( 4), which helps to flatten the ground state considerably and substantially lower the thiophene-thiophene rotation barrier. [Display omitted]

N,N′-dimethyl-2,7-diazapyrenium: a redox-dependent receptor for aromatic carboxylates

Studies are presented showing that N,N′-dimethyl-2,7-diazapyrenium, DAP 2+, acts as a redox-dependent receptor for aromatic carboxylates in aqueous solution. Cyclic voltammetric studies of DAP 2+ by itself indicate that its one-electron reduction to DAP + is only quasi-reversible in both aqueous and acetonitrile solutions due to the slow oxidation of DAP + back to DAP 2+. In aqueous solution, the oxidation is very sensitive to other conditions such as pH and the presence of small amounts of iodide. However, in pH 7 phosphate buffer, fairly reversible voltammetry is observed at slow scan rates. Under these conditions, addition of excess aromatic carboxylate results in large negative shifts in the E 1/2 of DAP 2+/+. Similar changes are observed in the 1H NMR spectrum of DAP 2+ in the presence of the different carboxylates, indicating that the potential shift is due to strong interactions between the carboxylates and DAP 2+. Upon reduction, the favorable coulombic interactions between the anionic carboxylates and the cationic pyrenium are weakened, resulting in the observed negative shifts in E 1/2. Although the perturbation of electrostatic interactions causes the potential shifts, the magnitude of the shifts correlates more with the strength of π-stacking interactions between the carboxylates and DAP 2+. Those carboxylates whose π system can come into maximum close contact with the DAP 2+ π system give the largest potential shifts.

A free energy analysis of nucleic acid base stacking in aqueous solution

This paper reports a theoretical study of the free energy contributions to nucleic acid base stacking in aqueous solution. Electrostatic interactions are treated by using the finite difference Poisson-Boltzmann method and nonpolar effects are treated with explicit calculation of van der Waals interactions and/or free energy-surface area relationships. Although for some pairs of bases there is a favorable Coulombic interaction in the stacked conformation, generally the net effect of electrostatic interactions is to oppose stacking. This result is caused by the loss of favorable base-solvent electrostatic interactions, that accompany the partial removal of polar atoms from water in the stacked conformation. Nonpolar interactions, involving the hydrophobic effect and enhancement of van der Waals interactions caused by close-packing, drive stacking. The calculations qualitatively reproduce the experimental dependence of stacking free energy on purine-pyrimidine composition.